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International Clinical Trials
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In the US, most labs generally use conventional urine culture. Twelve
local (or hospital) labs within the last year have been evaluated to see
if they can readily serve a complicated urinary tract infection (cUTI)
clinical trial.
- Seven labs routinely used a 1mcL loop for quantitative plate inoculation
- One used a 10mcL loop
- The rest used both 1mcL and 10mcL loops
- Six
out of 12 labs controlled colony counts – obtained by loop inoculation –
against plates inoculated with a calibrated pipette
- Six different labs had never performed quality control, or had performed it many years ago
- All labs were ready to undergo quality control for clinical trial purposes
As a result, only one lab was able to start serving clinical trials without any additional training and set-up.
In
European countries, along with a conventional quantitative technique, a
number of semi-quantitative culture methods are also used. An overview
of the most common is given in Table 1 (see full PDF). In Western
Europe, the majority of labs use a conventional quantitative technique –
although the details may vary – but some labs prefer other methods. For
instance, of eight laboratories (five hospitals and two commercial)
evaluated in Italy:
- Three labs performed quantitative plate inoculation with a 10mcL loop
- One carried out the procedure with a 10mcL and a 1mcL loop
- One used a 5mcL loop
- One used a 1mcL loop
- One utilised a Previ Isola system
- One used light scattering for determination of bacterial density in urine samples
In
contrast, most labs in Central and Eastern Europe employ
semi-quantitative culture. In former Soviet countries, hospital labs use
a semi-quantitative technique described in the Union of Soviet
Socialist Republics’ Ministry of Health Order, number 535. This method
has vague colony density interpretation criteria (see full PDF)).
Implementation of conventional quantitative technique in Eastern
European countries frequently requires additional supplies (such as
calibrated loops), training and additional supervision. In most cases,
hospital labs are not motivated to perform quantitative culture as it is
time-consuming, and do not provide additional diagnostic information as
compared to routinely used semiquantitative culture.
Commercial
labs in Georgia, Russia, Ukraine and so forth would usually consent to
implementation of conventional technique as per the American Society for
Microbiology for clinical trial purposes, although they also need
additional training and supervision; most labs further require a fee for
implementation of a method that is different from their familiar
system.
All methods are approved for diagnostic use as they
provide sufficient information for discerning between significant and
non-significant bacteriuria. However, these methods are all unable to
distinguish between <103 and >103 colony
forming units per millilitre (CFU/mL), so they cannot be used for
demonstration of microbiological success in trials with EMA submission.
Modern Methods
Having said this, some semi-quantitative methods (such as DipStreakTM or Previ Isola) might be acceptable for trials with FDA submission –
provided they give clear and traceable correlation between colony count
and CFU/mL.
Interpretation of bacterial growth with DiaSlide is
human factor-dependent; therefore, inclusion of a patient into modified
intention-to-treat (mITT) population, as well as into microbiologically
cured population, cannot be verified.
Previ Isola provides more explicit interpretation rules (for example, growth only in sector 1 means <104CFU/mL),
which makes the results less dependent on the human factor. Independent
validation has shown the agreement of semi-quantitative growth
estimation with the conventional manual plating (1). According to its
reference chart, Previ Isola differentiates <104CFU/mL and ≥104CFU/mL;
in other words, microbiological cure versus failure as per the FDA. On
the other hand, with Previ Isola, some patients eligible for mITT may be
lost, as its reference chart differentiates ≤105CFU/ mL versus >105CFU/mL – whereas according to FDA mITT, population is defined as patients having baseline pathogen growth ≥105CFU/mL.
DipStreak
provides correlation between the number of colonies per slide and
CFU/mL. Reporting intervals allow differentiation between mITT and
non-mITT, as well as between microbiological cure and failure as defined
by current FDA guidance. At the same time, this method is not able to
reliably demonstrate pathogen eradication for EMA submission. According
to the latest research, correlation between conventional quantitative
plating and DipStreak with CNA/McConkey agar is high (2).
In
most cases, Microbiology Manual by Central Lab provides a detailed
description of quantitative urine plating and colony count procedure.
However, experience shows that each microbiology lab should be carefully
evaluated for its ability and willingness to use a technique that is
different from their routine, and to perform quality control of colony
counts on loop-inoculated plates versus pipetteinoculated plates.
Colony Count Reporting
In
the clinical trial setting, lab reports are usually defined as source
documents for case report forms (CRFs). However, if quantitative urine
culture results appear in the report as rounded CFU/mL (104CFU/mL or 3x103CFU/mL,
for example), they cannot be regarded as a source but rather as an
interpretation of colony count. Source data is the exact number of
colonies per quantitative plate documented elsewhere (from one colony to
100 colonies; numbers above 100 are registered as >100).
Reporting
rounded CFU/mL can lead to inconsistencies between results obtained by
different labs, which may use a variety of rounding rules. One lab, for
instance, might report 60 colonies (1mcL loop) as ≥104CFU/mL, whereas another lab could report it as 105CFU/mL.
In
addition to different rounding rules, labs may establish varying
reporting intervals for CFU/mL. For example, some labs place round
colony counts with the smaller order of magnitude rather than with the
same: ≤103CFU/mL; >103 to ≤104CFU/mL;
and so on. The FDA’s definitions of baseline pathogen and
microbiological cure place round colony counts with the same order of
magnitude (pathogenic bacteria growth is ≥105CFU/mL, for example).
The
safest option to avoid the above discrepancies is reporting the number
of colonies (from one to 100 and >100) multiplied by the dilution
factor. This may not be easily achievable, however, as in most labs, the
reports are generated by laboratory information systems (LIS). In many
labs, LIS allows entry of rounded results only; in some cases, LIS may
perform rounding automatically using its own rules that cannot be
re-programmed for the trial purpose. Having said this, if the trial
needs are clearly explained to a laboratory during its evaluation and
set-up, the lab usually agrees to add the exact number of colonies in
the comment field of the report. Even if bacterial growth is captured in
the CRF in a rounded format, trained study monitors will be able to
re-check the data against raw colony counts available in the lab
reports.
Local versus Regional
Evaluation of
microbiology labs is a standard step in any antibiotic trial. A
project-specific questionnaire is developed for each study in order to
cover all specific logistics, testing and data management requirements.
In
countries where conventional quantitative culture is routinely used,
and overall performance and data management are of a high quality, it is
appropriate to use local (hospital) labs, as any gaps discovered during
their evaluation can be discussed and addressed in most cases. In a
cUTI trial carried out in the US and Germany, for example, local labs
were most frequently asked to use both 1mcL and 10mcL loops (as per
protocol); to provide the exact number of colonies in the report; and to
perform quality control checks of colony counts on loop-inoculated
plates versus pipetteinoculated plates.
In countries where
semi-quantitative culture is routinely used and overall quality of local
labs for clinical trials is questionable, the preferred choice is
regional (or commercial) labs. With preservative tubes, specimen
logistics is not an issue. Regional labs in countries like Bulgaria,
Romania, Russia and Ukraine have the following advantages:
- General management is of higher quality than at local labs (more reliable performance)
- No gaps in data management, which makes the results verifiable
- Higher flexibility in culture techniques and results reporting format
- Reliable isolate management
- Dedicated personnel to supervise the study
- Real time feedback on submitted samples quality, such as urine contamination
In
countries undergoing transit to EU laboratory standards (such as
Poland, Hungary or Slovenia), parallel evaluation of local and regional
labs may help to choose the optimal scenario.
From All Angles
Based
on past experience in cUTI clinical trials with microbiology labs
located in the US and Europe, the following conclusion can therefore be
drawn: to obtain uniform, reliable and verifiable pathogenic growth
results in a global cUTI trial, one should take into consideration the
country-specific features of different microbiological labs’ practices.
Failure to take these variations into account may lead to lost data due
to unnoticed ‘gaps’ and ‘bugs’ in culture techniques, reporting, quality
and data management.
References
1. Bustamante V,
Meza P, Román JC and García P, Evaluation of an automated streaking
system of urine samples for urine cultures, Rev Chilena Infectol 31(6):
pp670-675, December 2014
2. Colodner R and Keness Y, Evaluation of
DipStreak containing CNAMacConkey agar: A new bedside urine culture
device, Isr Med Assoc J 2(7): pp563-565, July 2000
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